Abstract

In natural history collections throughout Europe, there are many old lion specimens of
unknown origin. If these specimens can be shown to have originated from now-extinct
populations their value would significantly increase, as would the value of the collections.
Recently, a 200-year old mounted skeleton in the Zoological Museum Amsterdam has been
identified as the extinct Cape lion Panthera leo melanochaita (Smith, 1842),
based primarily on morphological information inferred from a painting of this specimen while
it was still alive. To test this hypothesis, we used ancient DNA (aDNA) techniques to
extract and sequence mitochondrial DNA (mtDNA) from this specimen, and compared the genetic
results with previously published lion mtDNA sequences. Our results show that the specimen
is not a Cape lion, but that it instead possesses the mtDNA haplotype of the Asiatic lions
P. l. persica (Meyer, 1826) from India. This Indian origin hypothesis is
further supported by an investigation of its cranial morphology. As the amount of genetic
information available for lions increases, in particular data from across their historic
distribution, the potential for aDNA techniques to identify the origins of previously
unassigned museum specimens continues to grow.

Introduction

The lion Panthera leo (Linnaeus, 1758) was once widespread throughout Africa and
southwestern Eurasia, ranging from South Africa to Greece, and from Morocco to India
(Nowell and Jackson, 1996). Most likely due to the expansion
of human populations, lions disappeared from Greece by c. 2000 years ago, and from Palestine
around the time of the Crusades c. 11th - 12th century. In the past 200 years, lions have
disappeared from many parts of their former range, including the southern part of South
Africa (by 1870), Turkey (1870), Tunisia (1891), Iraq (1918), Iran (1942), Morocco (1942)
and Algeria (by 1960) (Yamaguchi and Haddane 2002; Patterson,
2004; K. Difalla, personal communication). Among the now extinct populations two
are particularly famous: the North African Barbary lion P. l. leo
(Linnaeus, 1758) and the South African Cape lion P. l.
melanochaita (Smith, 1842) (Mazák 1975; Nowell and Jackson 1996). This range contraction has left fragmented
populations in sub-Saharan Africa with an estimated total population of c. 18,000 - 47,000,
and a single small population (c. 300) in India (Nowell and Jackson
1996; Bauer and Van der Merwe 2002; Chardonnet 2002).

Prior to their extinction, lions from North Africa, the Cape Province of South Africa,
Iraq, and several other sites were imported into Europe, where it was common for lions to be
exhibited in menageries and zoos (Guggisberg 1963; Hemmer 1978). After death, these specimens were deposited in natural
history museums or private collections, where they remain until today. Unfortunately, many
of these specimens are associated with little or no provenance information and this is
particularly true for specimens dating to the 19th century or earlier. As the recent range
contraction has resulted in the loss of a substantial part of the lion’s genetic diversity,
these European specimens represent a significant, as yet untapped resource for the
investigation of the evolution and historical distribution of lions. The recently
“discovered” 200-year old lion specimen at the Zoological Museum of the University of
Amsterdam, The Netherlands, is an important case in point (Van Bree,
1998). Previous analysis of this specimen has suggested that it is a Cape lion
(Van Bree, 1998). This identification relied heavily on a
painting made of this specimen while it was still alive by the Dutch artist P.G. van Os
(1776-1839) (Van Bree and Welman, 1996; Van
Bree, 1998; Reynaerts et al. 2006). However,
osteological evidence presented in Van Bree (1998) contradicts
this classification, as the measurements point out that this specimen possesses a large
interorbital breadth (the smallest distance between the orbits) relative to the postorbital
constriction (the smallest distance across the postorbital constriction), which is a typical
characteristic of the North African – Indian lion skulls, rather than sub-Saharan lions
(Hemmer 1974).

The ability to extract and analyse DNA sequences from old specimens provides a mechanism
for resolving this debate. Once an organism dies, enzymatic processes begin to break down
its DNA, however recent advances in molecular biological techniques have made it possible to
amplify short fragments of ancient DNA (aDNA), normally mitochondrial DNA (mtDNA), from
specimens up to 100,000 years old (Lindahl 1993). While lions
were among the first felids whose phylogeny was investigated using molecular methods
(O’Brien et al. 1987), genetic information has been lacking
for this species from many parts of its natural range. Indeed, until very recently,
published lion mt-DNA sequences originated from only seven countries, making up less than
half of its natural range: Botswana, India, Kenya, Namibia, South Africa, Tanzania, and
Uganda (Janczewski et al. 1995; Burger et al.
2004; Dubach et al. 2005). Under such
circumstances, even if mtDNA were successfully obtained from an unprovenanced specimen, it
would be difficult to determine its origin. However, Barnett et al.
(2006a, 2006b) amplified the HVR1 of mtDNA control
region from 32 modern lion specimens representing most of the extant and historical natural
range of lions. By comparing sequences obtained from unprovenanced museum specimens to the
increased data set, it may be possible to assign the origins of these specimens with
reasonable confidence.

In this paper, we re-examine the origin of the Amsterdam Cape lion using both aDNA
techniques and skull morphology analysis. We also discuss the feasibility of using these
techniques to identify previously unprovenanced museum specimens.

Material and methods

The interorbital breadth and the postorbital constriction were measured on the Amsterdam
“Cape” lion (ID number: ZMA710), and compared to those of lion skulls originated from India,
and the southern part of the former Cape Province, South Africa (Table
1). Classification was based on museum labels, and we also included into the
analysis a few old specimens labelled as Cape lion without any specific locations within
South Africa (see Table 1). We did not follow the
classification of the Cape lion suggested by Mazák (1975) which
was recently contradicted (Barnett et al., 2006a, 2006b).

A skull was classified as subadult if cemento-enamel junctions of all canines were already
visible above the alveoli of the cleaned skull and yet the basioccipital-basisphenoid
suture, and/or frontal suture, was still open. If those sutures were closed, a skull was
classified adult. Measurements were carried out using a set of metal calipers to the nearest
0.02mm. The coefficient of variation for the interorbital breadth was 0.06% (n= 5 skulls with 3 repeats each), and that for the postorbital constriction was
0.09% (n= 5 skulls with 3 repeats each). Following Yamaguchi et al. (2004) we arbitrarily accept a less than 2% coefficient of
variation as a cut-off line for reliability and consistency in measurements, which both met.

In addition to sexual size dimorphism that is common in the Felidae, it has been suggested
that the skull morphological characteristics of captive lions differ from those of wild
animals (Hollister, 1917). While both the painting and
morphometric data suggest that the Amsterdam Cape lion is a male, the sex of the specimen
was not explicitly given in the original record. Additionally, the specimen was known to
have spent at least some time in captivity (Van Bree, 1998). We
therefore included both male and female and both captive and wild individuals in our
comparative data set.

Table 1. Specimens of Cape lions and Asiatic lions that were analysed in
this study. Identification was based on museum labels. There may be more captive
specimens in addition to those that are clearly recorded as captive animals. The dates
indicate either when the specimens were collected (e.g. collected in the field, presented
to zoos, or died in zoos), or when the specimens were brought to the collections.
Abbreviations and the details of the collections are as followed: m (male), f (female), a
(adult), sa (subadult), London (Natural History Museum, London, UK), Oxford (Natural
History Museum, University of Oxford, Oxford, UK), Paris (National Museum of Natural
History, Paris, France), Leiden (National Natural History Museum, Leiden, The
Netherlands), Amsterdam (Zoological Museum, Amsterdam University, Amsterdam, The
Netherlands), Stockholm (Swedish Museum of Natural History, Stockholm, Sweden), Cape Town
(South African Museum, Cape Town, South Africa), KWT (Amathole Museum, King William’s
Town, South Africa), PE (Port Elizabeth Museum Complex, Port Elizabeth, South Africa), and
Bulawayo (Natural History Museum, Bulawayo, Zimbabwe). “Cape” is used here as the former
Cape Province, South Africa, which consists of the current Eastern Cape, Western Cape, and
Northern Cape provinces.

DNA extraction and analysis

Samples of a mounted lion skeleton (ID number: ZMA710) kept in the Zoological Museum of the
University of Amsterdam, The Netherlands, which was identified as the Cape lion (Van Bree, 1998), were obtained twice independently. First sampling was
carried out in 1999 (Sample-1), and the second in 2006 (Sample-2). Small pieces of dried
tissue (up to c. 10 × 10 × 5 mm) attached to the mounted skeleton were removed for DNA
analysis. Laboratory procedures were performed according to strict aDNA criteria following
Barnett et al. (2006a).

For both samples, a 130 base pair (bp) fragment of the mitochondrial hypervariable region
was amplified as described in Barnett et al (2006a). This
fragment was chosen as it is known to contain nucletotide polymorphisms that make it
possible to discriminate between lions originating from different populations.

Data authenticity

Sample-1 was extracted and amplified in July 2005 as part of a large-scale investigation of
the lion phylogenetics and evolution (for details see Barnett et al.
2006a, 2006b). Sample-2 was analysed in May 2006
following the procedure outlined in Barnett et al. (2006a).
Multiple PCR and extraction controls were also carried out for Sample-2, which was amplified
and cloned twice. All replications produced identical sequences. Sample-1 and Sample-2
produced identical sequences, and the sequences were deposited in GenBank (Accession number
EF517785).

The Felidae are known to contain macrosatellites (Numts) resulting from nuclear
translocation of the mtDNA (Lopez et al., 1997; Cracraft et al., 1998). Previous studies of Panthera mtDNA
control region have revealed a Numt significantly divergent from any mitochondrial copy
(Jae-Heup et al., 2001). Cloning of PCR products from
Sample-2 did not find any sequences with homology to the Panthera Numt. The
cloned mtDNA sequences obtained were then aligned with those of other lions (Barnett et al. 2006a, 2006b) using Se-Al
(Rambaut, 1996) and visually checked.

Results

The origin-known Asiatic lions from India were clearly separated from the origin-known Cape
lions based on the ratio between interorbital breadth and postorbital constriction (Fig. 1). The Amsterdam specimen was placed very close to the range for
the examined Indian lions, well outside of the range for the examined Cape lions (Fig. 1).

Approximately 130-bp of the mitochondrial DNA (HVR1) was amplified from both Sample-1 and
-2, and produced identical sequences. Comparison of this sequence with

Fig. 1. Scatterplot of Postorbital constriction against Interorbital breath of Indian
lions Panthera leo persica and Cape lions P. l. melanochaita as well
as the Amsterdam “Cape” lion, showing a clear distinction between the two subspecies. The
Amsterdam lion clusters with the Indian lions. Numbers are corresponding to those in
Table 1.

previously published data revealed them to be identical to sequences of the Asiatic lion
from India (Fig. 2).

Discussion

Our results based on both molecular and morphometric data, suggest that the specimen in
question likely belongs to the Indian population of the Asiatic lion rather than that of the
Cape lion, contradicting the previous identification by Van Bree
(1998).

Van Bree (1998) stated “…till the middle of the 19th century
the only lions arriving in Europe either came from North Africa or from the Cape.” He gives
no source for this assertion, but if this were true, the focal specimen, which died in 1809
(Van Bree, 1998), would most likely not have been an Asiatic
lion. However, natural history books published during the 18th - early 19th centuries
suggest that, while they were not common, Asiatic lions were imported into Europe before the
middle of the 19th century (Buffon and Daubenton, 1761; Bennett, 1829; Jardine, 1834). Because the
Cape Colony, South Africa, was under Dutch rule from 1652 until 1806, it is possible that
some lions imported to The Netherlands may have been Cape lions, and indeed Van Bree (1998), followed by Reynaerts et al.
(2006), used this line of reasoning to corroborate his conclusions. However, by
the end of the 18th century, the Dutch had established colonies and trading posts within or
bordering the range of the Asiatic lion in India, therefore it is also possible that lions
may have been exported to The Netherlands from these colonies. The most relevant of the
Dutch colonies in this region were a number of posts in Suratte, northwestern India,
opposite to the Kathiawar Peninsula, where the only remaining Indian lion population exists
today, and those in Coromandel on the east coast of India (Israel,
1990). Additionally, exotic animals were brought into and moved around Europe ever
since the

Fig. 3. Picture of a wild (bottom) and a captive (top) male Indian lion Panthera
leo persica, showing an example of phenotypic plasticity of the lion’s mane. The
photo of the wild lion was taken in the Gir Lion Sanctuary, north-west India, and that of
the captive in Berlin Zoo, Germany (photos: N. Yamaguchi).

Roman period, with a possible exception of the period between c. the 8th and 13th centuries
(Baratay and Hardouin-Fugier, 2002). In this context, even if
the Dutch had not possessed any trading post in India, Asiatic lions may have been imported
occasionally to The Netherlands.

Early authors believed that apparently distinct external morphological characters
particular to some lion populations, for example the male’s huge dark mane that extends
behind the shoulders and covers the ventral part of the body in Barbary and Cape lions,
could be used to identify lions as belonging to particular populations (Harper, 1945; Mazák, 1970, 1975). The identification of the Amsterdam specimen depended on the animal’s huge
dark mane as it was depicted in a painting of the animal when still alive (Van Bree, 1998). However, it is now known that size and colour of a
lion’s mane are influenced by various factors, including ambient temperature, animal’s age
and testosterone level (Kays and Patterson, 2002; West and Packer, 2002; Patterson et al.,
2006). In particular, the Asiatic lion from India, which is characterised by a
very small mane in its natural habitat, grows an enormous mane with its greater part being
dark or even black, which extends behind the shoulders and covers the belly, in European
zoos (Yamaguchi and Haddane, 2002; Yamaguchi unpublished, see
Fig. 3). Therefore, a heavy mane developed in a cooler place
(e.g. menageries and zoos in Europe) would be an inappropriate
marker (in comparison to molecular markers) for assigning the individuals to particular
populations.

The mtDNA control region (HVR1) has proven to be useful in constructing the general
intraspecific lion phylogeny, and has been used to identify molecular markers that are
capable of discriminating between dozens of lion populations throughout the species range
(Barnett et al. 2006a, 2006b).
Importantly, it appears that only a small fragment of the HVR1 (c. 130 bp) is required for
population-level identification: while genetic variation among lion mtDNA is low, the HVR1
is the most variable marker known, and contains as many informative sites as much longer
stretches of sequence in other regions (Barnett et al. 2006b).
This suggests that DNA identification of old museum specimens is not only realistic, but
also quick and cost-effective. The most significant difficulty associated with this approach
is that mtDNA, which is commonly used for aDNA analysis, is inherited strictly from the
mother, and therefore can only provide insight into the maternal lineage of the specimen
investigated. Techniques in aDNA analysis are advancing rapidly, however, and it may soon be
possible to isolate paternal-specific DNA sequences (e.g. Y-chromosome
DNA) from large numbers of specimens, which will enable identification of both the maternal
and paternal lineages. Such advances will provide significant insights into the historical
phylogenetics and behaviour of the lion (e.g. sexual difference in
their dispersal pattern), in addition to making possible the accurate assignation geographic
origin to poorly documented museum specimens. In spite of the difficulties associated with
aDNA research, it would be worthwhile for museums to re-examine old lion specimens of
unknown origin. Newly “discovered” specimens originated from extinct lion populations would
increase our understanding of these lost populations, which in turn would help us understand
the true diversity (e.g. morphological, anatomical, and genetic) of
the lion. In addition to those science- and conservation-oriented interests, specimens from
extinct lion populations would increase in value, and underscore the significance of museum
collections.